ProjectSummary Peptides are now widely used to treat human diseases and represent a fast-growing class of therapeutics in thebiopharmaceuticalmarket.Ingeneral,peptideshavetheadvantagesofhighspecificityandpotencybutthe disadvantages of being susceptible to aggregation and proteolytic degradation. Because of these drawbacks, therapeutic peptides are often administered through injections and, in order to maintain sufficient levels of circulating biologically active peptides, such injections are often frequent. This makes the treatment process expensive, inconvenient, and occasionally dangerous to patients, particularly during long-term treatment of chronicdiseases.Toaddresstheseissues,manyattemptshavebeenmadetodeveloplessinvasiveroutesfor peptide administration. By selectively retarding drug release in theacidic environmentof the stomach through the use of pH sensitive polymers, peptides can now be efficiently conveyed to the small intestine, where the majority of drug absorption occurs after oral delivery. The absorption of peptides in the small intestine is not, however, free of challenges. For example, self-association and degradation by proteases located in the small intestinallumencansignificantlylowerthebioavailabilityofpeptides. Recently, we have demonstrated that almost all of the relevant physical properties of peptides can be altered by attaching particular glycans to flexible or fragile regions. Based on these results, we hypothesize that better guidelines of using glycans in peptide engineering can be developed by deeply examining the effectsofglycosylationontworepresentativetherapeuticpeptides:humaninsulinandglucagon-likepeptide-1 (GLP-1). The objective of our proposed research is to test this hypothesis through chemical synthesis and biologicalcharacterizationofcollectionsofdifferentlyO-glycosylatedinsulinandGLP-1variants.Wewillbegin ourstudybyinvestigatinghowO-linkedglycansmodulatevariouspropertiesofinsulin.Thiswillbeachievedby designing, quantifying and then comparing the properties of a library of synthetically prepared, pure, and homogeneous insulin glycoforms with systematic variations in glycosylation patterns (glycosylation sites, glycan sizes and structures) and/or amino acid sequences. In the second part of the study, we will use a similarstrategytoevaluatetheeffectsofO-glycosylationonthepropertiesofGLP-1. Theproposedstudyisoneofthefirstattemptstodeveloparationalapproachtoglycoengineertherapeutic peptideswithafocusonO-glycosylation.Theresultsoftheproposedresearchareexpectedtoleadtoabetter understandingofpeptideglycoengineering,O-glycosylation,andfacilitatethedevelopmentofnoveltherapeutic peptidesforthetreatmentofhumandiseases.